Project Summary Down syndrome (DS), caused by trisomy for human chromosome 21 (Hsa21), is among the most complex genetic perturbations compatible with human survival past term; 80% of conceptuses with trisomy 21 miscarry. The interactions of ca. 600 transcribed sequences on Hsa21 affect cells directly and, by altering the actions of individual cells during development, via secondary effects on neighboring cells. The resulting outcomes comprise 80+ clinical features affecting every system in the body, although only a subset of these features is observed in any individual with trisomy 21. The advent of molecular genetics in DS research brought with it single gene transgenic mice made with newly discovered Hsa21 genes; phenotypes were interpreted from the reductionist perspective that the DS phenotype is the sum of independently acting single gene effects. Davisson?s Ts65Dn mouse fundamentally changed the paradigm for DS research, providing a model trisomic for a large subset of Hsa21 orthologs. Analysis of these mice showed that they manifest trisomy in ways that are comparable to some outcomes of trisomy 21 in people. Ts65Dn has been the work horse for more than two decades, however, advances such as the genome project show weaknesses in that model. Given the critical role for the mouse in developing therapies to ameliorate features of DS, better genetic models are needed. We will characterize a newly established model, ?MAC21?, carrying Hsa21 as a mouse artificial chromosome. It contains a better genetic representation of Hsa21 than any extant model and unlike other models with Hsa21, these mice are not mosaic. Thus MAC21 avoids many limitations of existing DS models (Specific Aim 1). While all effects of trisomy are ultimately a product of misexpression in specific cells, we can usefully differentiate trisomic phenotypes that are a product of maldevelopment from those which represent an ongoing impact of trisomy on cell function. For example, midface skeletal retrusion that is substantially responsible for the characteristic facial appearance of people with DS and Ts65Dn mice arises due to a reduction of cranial neural crest cells at all stages of delamination from the neural tube, migration and proliferation that forms the first pharyngeal arch, thus there are too few cells to properly form Meckel?s cartilage. Hence the ?flat? skeletal aspect is due to hypocellularity as a consequence of a developmental anomaly due to trisomy, while the cells composing these structures appear to be function normally. Similarly, hypoplastic cerebellum in DS and Ts65Dn is a product of inadequate proliferation of granule cell precursors, but function of adult cells appears largely normal. Using CRISPR-based ablation of Hsa21 in specific cells at specific stages, we will seek to differentiate these effects. The distinction may seem academic, but in fact, specific knowledge of the timing as well as the nature of DS phenotypes is critical to the successful application of animal models in the development of prenatal therapies for complex genetic disease (Specific Aim 2).